Methods for the early diagnosis of ovarian cancer

ABSTRACT

The present invention discloses the protease stratum corneum chymotryptic enzyme (SCCE) is specifically over-expressed in ovarian and other malignancies. A number of SCCE peptides can induce immune responses to SCCE, thereby demonstrating the potential of these peptides in monitoring and the development of immunotherapies for ovarian and other malignancies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. Ser. No. 10/372,521, filed Feb.21, 2003, which claims benefit of priority under 35 USC §120 ofcontinuation-in-part application U.S. Ser. No. 09/918,243, filed Jul.30, 2001, now U.S. Pat. No. 6,627,403, which is a continuation-in-partof U.S. Ser. No. 09/905,083, filed Jul. 13, 2001, which is a divisionalof U.S. Ser. No. 09/502,600, now U.S. Pat. No. 6,294,344, which is acontinuation-in-part of U.S. Ser. No. 09/039,211, filed Mar. 14, 1998,now U.S. Pat. No. 6,202,318, which claims benefit of provisional U.S.Ser. No. 60/041,404, filed Mar. 19, 1997, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Generally, the present invention relates to the fields of molecularbiology and medicine. More specifically, the present invention is in thefield of cancer research, especially ovarian cancer diagnosis.

2. Description of the Related Art

In order for malignant cells to grow, spread or metastasize, they musthave the capacity to invade local host tissue, dissociate or shed fromthe primary tumor, enter and survive in the bloodstream, implant byinvasion into the surface of the target organ and establish anenvironment conducive for new colony growth, including the induction ofangiogenic and growth factors. During this progression, natural tissuebarriers such as basement membranes and connective tissue have to bedegraded. These barriers include collagen, laminin, fibronectin,proteoglycans and extracellular matrix glycoproteins. Degradation ofthese natural barriers, both those surrounding the primary tumor and atthe sites of metastatic invasion, is believed to be brought about by theaction of a matrix of extracellular proteases.

Proteases have been classified into four families: serine proteases,metallo-proteases, aspartic proteases and cysteine proteases. Manyproteases have been shown to be involved in human disease processes andthese enzymes are targets for the development of inhibitors as newtherapeutic agents. Certain individual proteases are induced andoverexpressed in a diverse group of cancers, and as such, are potentialcandidates for markers of early diagnosis and targets for possibletherapeutic intervention. A group of known protease expressed in variouscancers are shown in Table 1.

TABLE 1 Proteases Gastric Brain Breast Ovarian Serine uPA uPA NES-1NES-1 PAI-1 PAI-1 uPA uPA tPA PAI-2 Cysteine: CatSCCE B CatSCCE LCatSCCE B CatSCCE B CatSCCE L CatSCCE L CatSCCE L Metallo- Matrilysin*Matrilysin Stromelysin-3 MMP-2 Collagenase* Stromelysin MMP-8Stromelysin-1* Gelatinase B MMP-9 Gelatinase A uPA, Urokinase-typeplasminogen activator; tPA, Tissue-type plasminogen activator; PAI-I,Plasminogen activator 0 inhibitors; PAI-2, Plasminogen activatorinhibitors; NES-1, Normal epithelial cell-specific-1; MMP, Matrix Pmetallo-protease. *Overexpressed in gastrointestinal ulcers.

There is a good body of evidence supporting the downregulation orinhibition of individual proteases and the reduction in invasivecapacity or malignancy. In work by Clark et al., inhibition of in vitrogrowth of human small cell lung cancer was demonstrated using a generalserine protease inhibitor. More recently, Torres-Rosedo et al. (1993)demonstrated an inhibition of hepatoma tumor cell growth using specificantisense inhibitors for the serine protease hepsin. Metastaticpotential of melanoma cells has also been shown to be reduced in a mousemodel using a synthetic inhibitor, batimastat, of metallo-proteases.Powell et al. (1993) presented evidence to confirm that the expressionof extracellular proteases in a non-metastatic prostate cancer cell lineenhances their malignant progression. Specifically, enhanced metastasiswas demonstrated after introducing and expressing the PUMP-1metallo-protease gene. There is also a body of data to support thenotion that expression of cell surface proteases on relativelynon-metastatic cell types increases the invasive potential of suchcells.

To date, ovarian cancer remains the number one killer of women withgynecologic malignant hyperplasia. Approximately 75% of women diagnosedwith such cancers are already at an advanced stage (III and IV) of thedisease at their initial diagnosis. During the past 20 years, neitherdiagnosis nor five-year survival rates have greatly improved for thesepatients. This is substantially due to the high percentage of high-stageinitial detection of the disease. Therefore, the challenge remains todevelop new markers that improve early diagnosis and thereby reduce thepercentage of high-stage initial diagnoses. The ability to disengagefrom one tissue and re-engage the surface of another tissue is whatprovides for the morbidity and mortality associated with this disease.Therefore, extracellular proteases may be good candidates for markers ofmalignant ovarian hyperplasia.

Thus, the prior art is deficient in a tumor marker useful as anindicator of early disease, particularly for ovarian cancers. Thepresent invention fulfills this long-standing need and desire in theart.

SUMMARY OF THE INVENTION

This invention allows for the detection of cancer, especially ovariancancer, by screening for stratum corneum chymotryptic enzyme (SCCE) mRNAin tissue. Stratum corneum chymotryptic enzyme specifically associateswith the surface of 80 percent of ovarian and other tumors. Proteasesare considered to be an integral part of tumor growth and metastasis,and therefore, markers indicative of their presence or absence areuseful for the diagnosis of cancer. Furthermore, the present inventionis useful for treatment, i.e., by inhibiting SCCE or expression of SCCE,for targeted therapy, for vaccination, etc.

The present invention provides methods of vaccinating an individualagainst SCCE or produce immune-activated cells directed toward SCCE byinoculating an individual with an expression vector encoding a SCCEprotein or a fragment thereof.

The present invention also provides methods of immunotherapy targetedtoward SCCE in an individual, involving the steps of generatingdendritic cells in vitro from peripheral blood drawn from an individual,loading these dendritic cells with SCCE protein or a fragment thereof,then transferring these dendritic cells back to the individual in singleor multiple doses. SCCE-loaded or SCCE-expressing dendritic cells canalso be used to stimulate SCCE-specific T cell responses in vitro,followed by adoptive immunotherapy in which the individual is givenautologous SCCE-specific T cells.

There is also provided a method of monitoring the efficacy ofvaccinating an individual with SCCE or SCCE peptide. The methodcomprises measuring immune responses in response to said SCCE or SCCEpeptide, wherein induction of immune responses to said SCCE or SCCEpeptide indicates that said individual has been vaccinated against SCCE.

In another embodiment of the present invention, there are providedmethods of inhibiting expression of SCCE in a cell by introducing into acell a vector encoding an antisense SCCE RNA or an antibody that bindsthe SCCE protein.

In yet another embodiment of the present invention, there is provided amethod of targeted therapy to an individual using a compound that has atargeting moiety specific for SCCE and a therapeutic moiety.

In still yet another embodiment of the present invention, there areprovided compositions comprising immunogenic fragments of SCCE proteinor an oligonucleotide having a sequence complementary to SEQ ID No: 30.Also embodied is a method of treating a neoplastic state in anindividual with an effective dose of the above-describedoligonucleotide.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings have been included herein so that theabove-recited features, advantages and objects of the invention willbecome clear and can be understood in detail. These drawings form a partof the specification. It is to be noted, however, that the appendeddrawings illustrate preferred embodiments of the invention and shouldnot be considered to limit the scope of the invention.

FIG. 1 shows agarose gel comparison of PCR products derived from normaland carcinoma cDNA.

FIG. 2 shows Northern blot analysis of ovarian tumors using SCCE, SCCE,PUMP-1, TADG-14 and β-tubulin probes.

FIG. 3 shows amplification with serine protease redundant primers:histidine sense (S1) with aspartic acid antisense (AS1), using normalcDNA (Lane 1) and tumor cDNA (Lane 2); and histidine sense (S1) withserine antisense (AS2), using normal cDNA (Lane 3) and tumor cDNA (Lane4).

FIG. 4 shows amplification with cysteine protease redundant primers.Normal (Lane 1), low malignant potential (Lane 2), serious carcinoma(Lane 3), mucinous carcinoma (Lane 4), and clear cell carcinoma (Lane5).

FIG. 5 shows amplification with metallo-protease redundant primers.Normal (Lane 1), low malignant potential (Lane 2), serious carcinoma(Lane 3), mucinous carcinoma (Lane 4), and clear cell carcinoma (Lane5).

FIG. 6 shows quantitative PCR analysis of SCCE expression. Cases 3, 4and 9 are normal ovaries. Cases 19, 21, 14, 15 and 16 are LMP tumors.Cases 43, 23, 36 and 37 are ovarian carcinomas. Expression levels ofstratum corneum chymotryptic enzyme relative to β-tubulin aresignificantly elevated in tumor Cases 19, 14, 15, 16, 43, 23, 36 and 37compared to that of normal ovaries.

FIG. 7A shows Northern blot analysis of stratum corneum chymotrypticenzyme mRNA from normal ovary and ovarian carcinomas. Lane 1, normalovary (case 10); Lane 2, serous carcinoma (case 35); Lane 3, mucinouscarcinoma (case 48); Lane 4, endometrioid carcinoma (case 51); and Lane5, clear cell carcinoma (case 54). Two transcripts (1.2 and 2.0 kb) weredetected in all of the subtypes of carcinoma (lanes 2-5). FIGS. 7B and7C show that normal human adult tissues (spleen, thymus, prostate,testis, ovary, small intestine, colon, peripheral blood leukocyte,heart, brain, placenta, lung, liver, skeletal muscle, kidney andpancreas) and normal human fetal tissues (brain, lung, liver and kidney)examined showed no visible SCCE transcripts.

FIG. 8 shows the ratio of SCCE expression to expression of β-tubulin innormal ovary, LMP tumor and ovarian carcinoma. SCCE mRNA expressionlevels were significantly elevated in LMP tumor (p<0.05) and carcinoma(p<0.001) compared to that in normal ovary. All 10 cases of normalovaries showed a low level of SCCE mRNA expression.

FIG. 9 shows MDA-MB-435S (Lanes 1 & 3) and HeLa (Lanes 2 & 4) celllysates were separated by SDS-PAGE and immunoblotted. Lanes 1 & 2 wereprobed with rabbit pre-immune serum as a negative control. Lanes 3 & 4were probed with polyclonal rabbit antibodies generated to peptidesderived from SCCE protein sequence.

FIG. 10A shows normal surface ovarian epithelium. Little SCCE expressionwas observed (normal ovary, X100). FIG. 10B is a negative controlsection for FIG. 10A. No nonspecific staining was observed (Normalovary, X100). FIG. 10C shows positive SCCE staining localized in thecytoplasm and the cell membrane of ovarian cancer cells (case 947, clearcell adenocarcinoma, X100). FIG. 10D is a negative control section forFIG. 10C. No nonspecific staining was observed (case 947, clear celladenocarcinoma, X100). FIG. 10E is positive stratum corneum chymotrypticenzyme staining localized in the cytoplasm and the cell membrane ofovarian cancer cells. Mucin in the glands also showed positive stratumcorneum chymotryptic enzyme staining (case 947, clear celladenocarcinoma, X100). FIG. 10F is a negative control section for FIG.10E. No nonspecific staining was observed (case 947, clear celladenocarcinoma, X100).

FIG. 11A shows Northern blot analysis of hepsin expression in normalovary and ovarian carcinomas. Lane 1, normal ovary (case 10); lane 2,serous carcinoma (case 35); lane 3, mucinous carcinoma (case 48); lane4, endometrioid carcinoma (case 51); and lane 5, clear cell carcinoma(case 54). In cases 35, 51 and 54, more than a 10-fold increase in thehepsin 1.8 kb transcript abundance was observed. Northern blot analysisof hepsin in normal human fetal (FIG. 11B) and adult tissues (FIG. 11C).Significant overexpression of the hepsin transcript is noted in bothfetal liver and fetal kidney. Notably, hepsin overexpression is notobserved in normal adult tissue. Slight expression above the backgroundlevel is observed in the adult prostate.

FIG. 12A shows hepsin expression in normal (N), mucinous (M) and serous(S) low malignant potential (LMP) tumors and carcinomas (CA). FIG. 12Bshows a bar graph of expression of hepsin in 10 normal ovaries and 44ovarian carcinoma samples. 0:<Mean+2SD; i:>Mean+2SD<4SD; 2::>Mean+4SD.

FIG. 13 shows a comparison by quantitative PCR of normal and ovariancarcinoma expression of mRNA for protease M.

FIG. 14 shows TADG-12 catalytic domain with an insert near the His5′-end.

FIG. 15A shows northern blot analysis comparing TADG-14 expression innormal and ovarian carcinoma tissues. FIG. 15B shows preliminaryquantitative PCR amplification of normal and carcinoma cDNAs usingspecific primers for TADG-14.

FIG. 16A shows northern blot analysis of the PUMP-1 gene in normal ovaryand ovarian carcinomas. FIG. 16B shows northern blot analysis of thePUMP-1 gene in human fetal tissue. FIG. 16C shows northern blot analysisof the PUMP-1 gene in adult tissues.

FIG. 17A shows a comparison of PUMP-1 expression in normal and carcinomatissues using quantitative PCR with an internal β-tubulin control. FIG.17B shows the ratio of mRNA expression of PUMP-1 compared to theinternal control β-tubulin in 10 normal and 44 ovarian carcinomas. SD isas in FIG. 12B.

FIG. 18 shows a comparison of Cathepsin L expression in normal andcarcinoma tissues using quantitative PCR with an internal β-tubulincontrol.

FIG. 19 is a summary of PCR amplified products for the hepsin, SCCE,protease M, PUMP-1 and Cathepsin L genes.

FIG. 20 shows CD8⁺ CTL recognition of SCCE 5-13 peptide in a 5 hr ⁵¹Crrelease assay. Targets were LCL loaded with SCCE 5-13 () and controlLCL (◯).

FIG. 21 shows CD8⁺ CTL recognition of SCCE 123-131 peptide in a 5 hr⁵¹Cr release assay. Targets were LCL loaded with SCCE 123-131 () andcontrol LCL (◯).

FIG. 22 shows peptide-specific CD8⁺ CTL recognition of endogenouslyprocessed and presented SCCE tumor antigen. CTL were derived bystimulation with dendritic cells pulsed with SCCE peptide 123-131.Cytotoxicity was tested in a standard 5 hours ⁵¹Cr-release assay againstautologous macrophages infected with Ad-GFP/SCCE (♦), macrophagesinfected with Ad-GFP-hepsin (▴), macrophages pulsed with SCCE 123-131peptide (▪), or control untreated macrophages ().

FIG. 23 shows CD8⁺ CTL specific for SCCE 123-131 lyse ovarian tumorcells. Target cells are autologous LCL loaded with 5 mg/ml peptide (♦),control LCL (open diamond), CaOV-3 ovarian tumor cells (▴), CaOV-3 tumorcells plus 1/25 dilution of BB7.2 ascites (▪), CaOV-3 ovarian tumorcells plus 50 mg/ml W6/32(), and K562 cells (◯).

DETAILED DESCRIPTION OF THE INVENTION

This invention identifies stratum corneum chymotryptic enzyme (SCCE) asa marker for ovarian tumor cells. In various combinations with otherproteases, stratum corneum chymotryptic enzyme expression ischaracteristic of individual tumor types. Such information can providethe basis for diagnostic tests (assays or immunohistochemistry) andprognostic evaluation (depending on the display pattern).

Long-term treatment of tumor growth, invasion and metastasis has notsucceeded with existing chemotherapeutic agents. Most tumors becomeresistant to drugs after multiple cycles of chemotherapy. The presentinvention identifies SCCE as a new therapeutic intervention targetutilizing either antibodies directed at the protease, antisense vehiclesfor downregulation or protease inhibitors both from establishedinhibition data and/or for the design of new drugs.

The present invention provides a method of vaccinating an individualagainst SCCE, comprising the steps of inoculating an individual with anexpression vector encoding a SCCE peptide or with peptide-loadeddendritic cells. Expression of the SCCE peptide elicits an immuneresponse in the individual, thereby vaccinating the individual againstSCCE. Generally, this method is applicable when the individual hascancer or is at risk of getting a cancer such as ovarian cancer, lungcancer, prostate cancer, pancreatic cancer and colon cancer. Sequencesof preferred SCCE peptides are shown in SEQ ID NOS: 31, 32, 33, 34, 35,36, 80, 86 and 99.

The present invention also provides a method of producingimmune-activated cells directed toward SCCE, comprising the steps ofexposing immune cells to SCCE protein or fragment thereof. Typically,exposure to SCCE protein or fragment thereof activates the immune cells,thereby producing immune-activated cells directed toward SCCE.Generally, the immune-activated cells are B-cells, T-cells and/ordendritic cells. Preferably, the SCCE fragment is a 9-residue fragmentup to a 20-residue fragment, and more preferably, the fragment is SEQ IDNOS: 31, 32, 33, 34, 35, 36, 80, 86 and 99. Oftentimes, the dendriticcells are isolated from an individual prior to exposure and thenreintroduced into the individual subsequent to the exposure. Typically,the individual has cancer or is at risk of getting a cancer such asovarian cancer, lung cancer, prostate cancer and colon cancer.

The present invention also provides methods of immunotherapy targetedtoward SCCE in an individual. In one embodiment, the method involvesgenerating dendritic cells in vitro from peripheral blood drawn from theindividual, loading these dendritic cells with SCCE protein or afragment thereof by lipofection or other means, then transferring thesedendritic cells back to the individual in single or multiple doses. SCCEmay also be expressed in these dendritic cells following transductionwith a recombinant DNA vector. Alternatively, SCCE-loaded orSCCE-expressing dendritic cells can be used to stimulate SCCE-specific Tcell responses in vitro, followed by adoptive immunotherapy in which theindividual is given autologous SCCE-specific T cells. Typically, theindividual has cancer or is at risk of getting a cancer such as ovariancancer, lung cancer, prostate cancer, pancreatic cancer and coloncancer. In general, a full length or a fragment of SCCE protein isexpressed in the isolated dendritic cells. Preferably, the fragment is a9-residue fragment up to a 20-residue fragment, and more preferably, thefragment is SEQ ID NOS: 31, 32, 33, 34, 35, 36, 80, 86 and 99.

There is also provided a method of monitoring the efficacy ofvaccinating an individual with SCCE or SCCE peptide such as those shownin SEQ ID NOS: 31, 32, 33, 34, 35, 36, 80, 86 and 99. The methodcomprises isolating T cells or CD8⁺ T cells from the vaccinatedindividual and measuring immune responses specific to the SCCE or SCCEpeptide. An increased level of immune responses compared to thoseexhibited by cells from normal individual indicates that said individualhas been vaccinated by the SCCE or SCCE peptide. In general, theindividual is vaccinated to SCCE if there is an increased level ofSCCE-specific T cells proliferation, an increased frequency ofSCCE-specific T cells or an increased frequency of SCCE-specificcytokine-secreting T cells. Standard assays well-known in the art suchas tetramer analysis and ELISPOT assay can be used to determine thefrequency of SCCE-specific T cells and the frequency of SCCE-specificcytokine-secreting T cells respectively.

In another aspect of the present invention, there is provided a methodof inhibiting expression of SCCE in a cell, comprising the step ofintroducing into a cell a vector comprises a sequence complementary toSEQ ID NO: 30, wherein expression of the vector produces SCCE antisenseRNA in the cell. The SCCE antisense RNA hybridizes to endogenous SCCEmRNA, thereby inhibiting expression of SCCE in the cell.

Expression of SCCE can also be inhibited by antibody. An antibodyspecific for a SCCE protein or a fragment thereof is introduced into acell, and binding of the antibody to SCCE would inhibit the SCCEprotein. Preferably, the SCCE fragment is a 9-residue fragment up to a20-residue fragment, and more preferably, the fragment is SEQ ID NOS:31, 32, 33, 34, 35, 36, 80, 86 and 99.

The present invention is also directed toward a method of targetedtherapy to an individual, comprising the step of administering acompound to an individual, wherein the compound has a targeting moietyspecific for SCCE and a therapeutic moiety. Preferably, the targetingmoiety is an antibody specific for SCCE, a ligand or ligand bindingdomain that binds SCCE. Likewise, the therapeutic moiety is preferably aradioisotope, a toxin, a chemotherapeutic agent, an immune stimulant orcytotoxic agent. Generally, the individual suffers from a disease suchas ovarian cancer, lung cancer, prostate cancer, colon cancer,pancreatic cancer or another cancer in which SCCE is overexpressed.

The present invention is further directed toward an immunogeniccomposition, comprising an appropriate adjuvant and an immunogenic fulllength SCCE protein or a fragment thereof. Preferably, the fragment is a9-residue fragment up to a 20-residue fragment, and more preferably, thefragment is SEQ ID NOS: 31, 32, 33, 34, 35, 36, 80, 86 and 99.

The present invention also provides an oligonucleotide having a sequencecomplementary to SEQ ID NO: 30 or a fragment thereof. The presentinvention further provides a composition comprising the above-describedoligonucleotide and a physiologically acceptable carrier, and a methodof treating a neoplastic state in an individual, comprising the step ofadministering to the individual an effective dose of the above-describedoligonucleotide. Typically, the neoplastic state may be ovarian cancer,breast cancer, lung cancer, colon cancer, prostate cancer, pancreaticcancer or another cancer in which SCCE is overexpressed.

It will be apparent to one skilled in the art that various substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art.

Such techniques are explained fully in the literature. See, e.g.,Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual(1982); “DNA Cloning: A Practical Approach,” Volumes I and II (D. N.Glover ed. 1985); “Oligonucleotide Synthesis” (M. J. Gait ed. 1984);“Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins eds. 1985);“Transcription and Translation” (B. D. Hames & S. J. Higgins eds. 1984);“Animal Cell Culture” (R. I. Freshney, ed. 1986); “Immobilized Cells AndEnzymes” (IRL Press, 1986); B. Perbal, “A Practical Guide To MolecularCloning” (1984). Therefore, if appearing herein, the following termsshall have the definitions set out below.

As used herein, the term “cDNA” shall refer to the DNA copy of the mRNAtranscript of a gene.

As used herein, the term “PCR” refers to the polymerase chain reactionthat is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis,as well as other improvements now known in the art.

The present invention comprises a vector comprising a DNA sequence whichencodes a SCCE protein, wherein said vector is capable of replication ina host, and comprises, in operable linkage: a) an origin of replication;b) a promoter; and c) a DNA sequence coding for said SCCE protein.Preferably, the vector of the present invention contains a portion ofthe DNA sequence shown in SEQ ID NO: 30. Vectors may be used to amplifyand/or express nucleic acid encoding a SCCE protein, a fragment of SCCEprotein, or an antisense SCCE mRNA.

An expression vector is a replicable construct in which a nucleic acidsequence encoding a polypeptide is operably linked to suitable controlsequences capable of effecting expression of the polypeptide in a cell.The need for such control sequences will vary depending upon the cellselected and the transformation method chosen. Generally, controlsequences include a transcriptional promoter and/or enhancer, suitablemRNA ribosomal binding sites and sequences which control the terminationof transcription and translation. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining appropriate transcriptional and translational controlsignals. See, for example, techniques described in Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold SpringHarbor Press, N.Y. A gene and its transcription control sequences aredefined as being “operably linked” if the transcription controlsequences effectively control transcription of the gene. Vectors of theinvention include, but are not limited to, plasmid vectors and viralvectors. Preferred viral vectors of the invention are those derived fromretroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpesviruses.

As used herein, the term “host” is meant to include not only prokaryotesbut also eukaryotes such as yeast, plant and animal cells. A recombinantDNA molecule or gene which encodes a human SCCE protein of the presentinvention can be used to transform a host using any of the techniquescommonly known to those of ordinary skill in the art. Especiallypreferred is the use of a vector containing coding sequences for thegene which encodes a human SCCE protein of the present invention forpurposes of prokaryote transformation. Prokaryotic hosts may include E.coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis.Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cellsand insect cells.

The term “oligonucleotide”, as used herein, is defined as a moleculecomprised of two or more ribonucleotides, preferably more than three.Its exact size will depend upon many factors, which, in turn, dependupon the ultimate function and use of the oligonucleotide. The term“primer”, as used herein, refers to an oligonucleotide, whetheroccurring naturally (as in a purified restriction digest) or producedsynthetically, and which is capable of initiating synthesis of a strandcomplementary to a nucleic acid when placed under appropriateconditions, i.e., in the presence of nucleotides and an inducing agent,such as a DNA polymerase, and at a suitable temperature and pH. Theprimer may be either single-stranded or double-stranded and must besufficiently long to prime the synthesis of the desired extensionproduct in the presence of the inducing agent. The exact length of theprimer will depend upon many factors, including temperature, sequenceand/or homology of primer and the method used. For example, indiagnostic applications, the oligonucleotide primer typically contains15-25 or more nucleotides, depending upon the complexity of the targetsequence, although it may contain fewer nucleotides.

The primers herein are selected to be “substantially” complementary toparticular target DNA sequences. This means that the primers must besufficiently complementary to hybridize with their respective strands.Therefore, the primer sequence need not reflect the exact sequence ofthe template. For example, a non-complementary nucleotide fragment,i.e., containing a restriction site, may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementary with the sequence tohybridize therewith and form the template for synthesis of the extensionproduct.

As used herein, “substantially pure DNA” means DNA that is not part of amilieu in which the DNA naturally occurs, by virtue of separation(partial or total purification) of some or all of the molecules of thatmilieu, or by virtue of alteration of sequences that flank the claimedDNA. The term therefore includes, for example, a recombinant DNA whichis incorporated into a vector, into an autonomously replicating plasmidor virus, or into the genomic DNA of a prokaryote or eukaryote; or whichexists as a separate molecule, e.g., a cDNA or a genomic or cDNAfragment produced by polymerase chain reaction (PCR) or restrictionendonuclease digestion, independent of other sequences. It also includesa recombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence, e.g., a fusion protein. Also included is arecombinant DNA which includes a portion of the nucleotides listed inSEQ ID NO: 30 and which encodes an alternative splice variant of SCCE.

The DNA may have at least about 70% sequence identity to the codingsequence of the nucleotides listed in SEQ ID NO: 30, preferably at least75%, e.g., at least 80%; and most preferably at least 90%. The identitybetween two sequences is a direct function of the number of matching oridentical positions. When a position in both of the two sequences isoccupied by the same monomeric subunit, e.g., if a given position isoccupied by an adenine in each of two DNA molecules, then they areidentical at that position. For example, if 7 positions in a sequence 10nucleotides in length are identical to the corresponding positions in asecond 10-nucleotide sequence, then the two sequences have 70% sequenceidentity. The length of comparison sequences will generally be at least50 nucleotides, preferably at least 60 nucleotides, more preferably atleast 75 nucleotides, and most preferably 100 nucleotides. Sequenceidentity is typically measured using sequence analysis software(Sequence Analysis Software Package of the Genetics Computer Group(GCG), University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705).

Further included in this invention are SCCE proteins which are encoded,at least in part, by portions of SEQ ID NO: 30, e.g., products ofalternative mRNA splicing or alternative protein processing events, orin which a section of SCCE sequence has been deleted. The fragment, orthe intact SCCE polypeptide, may be covalently linked to anotherpolypeptide, e.g., one which acts as a label, a ligand or a means toincrease antigenicity.

A substantially pure SCCE protein may be obtained, for example, byextraction from a natural source; by expression of a recombinant nucleicacid encoding a SCCE polypeptide; or by chemically synthesizing theprotein. Purity can be measured by any appropriate method, e.g., columnchromatography, such as immunoaffinity chromatography using an antibodyspecific for SCCE, polyacrylamide gel electrophoresis, or HPLC analysis.A protein is substantially free of naturally associated components whenit is separated from at least some of those contaminants which accompanyit in its natural state. Thus, a protein which is chemically synthesizedor produced in a cellular system different from the cell from which itnaturally originates will be, by definition, substantially free from itsnaturally associated components. Accordingly, substantially pureproteins include eukaryotic proteins synthesized in E. coli, otherprokaryotes, or any other organism in which they do not naturally occur.

In addition to substantially full-length proteins, the invention alsoincludes fragments, e.g., antigenic fragments, of the SCCE protein. Asused herein, “fragment,” as applied to a polypeptide, will ordinarily beat least 10 residues, more typically at least 20 residues, andpreferably at least 30, e.g., 50, residues in length, but less than theentire, intact sequence. Fragments of the SCCE protein can be generatedby methods known to those skilled in the art, e.g., by enzymaticdigestion of naturally occurring or recombinant SCCE protein, byrecombinant DNA techniques using an expression vector that encodes adefined fragment of SCCE, or by chemical synthesis. The ability of acandidate fragment to exhibit a characteristic of SCCE, e.g., binding toan antibody specific for SCCE, can be assessed by methods known in theart.

Purified SCCE or antigenic fragments of SCCE can be used to generate newantibodies or to test existing antibodies, e.g., as positive controls ina diagnostic assay) by employing standard protocols known to thoseskilled in the art. Included in this invention is polyclonal antiseragenerated by using SCCE or a fragment of SCCE as the immunogen in, e.g.,rabbits. Standard protocols for monoclonal and polyclonal antibodyproduction known to those skilled in this art are employed. Themonoclonal antibodies generated by this procedure can be screened forthe ability to identify recombinant SCCE cDNA clones, and to distinguishthem from other cDNA clones.

The invention encompasses not only an intact anti-SCCE monoclonalantibody, but also an immunologically-active antibody fragment, e.g., aFab or (Fab)₂ fragment; an engineered single chain Fv molecule; or achimeric molecule, e.g., an antibody which contains the bindingspecificity of one antibody, e.g., of murine origin, and the remainingportions of another antibody, e.g., of human origin.

In one embodiment, the antibody, or a fragment thereof, may be linked toa toxin or to a detectable label, e.g., a radioactive label,non-radioactive isotopic label, fluorescent label, chemiluminescentlabel, paramagnetic label, enzyme label, or colorimetric labelwell-known in the art. Examples of suitable toxins include diphtheriatoxin, Pseudomonas exotoxin A, ricin, and cholera toxin. Examples ofsuitable enzyme labels include alkaline phosphatase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, etc.Examples of suitable radioisotopic labels include ³H, ¹²⁵I, ¹³¹I, ³²P,³⁵S, ¹⁴C, etc.

Paramagnetic isotopes for purposes of in vivo diagnosis can also be usedaccording to the methods of this invention. There are numerous examplesof elements that are useful in magnetic resonance imaging. Fordiscussions on in vivo nuclear magnetic resonance imaging, see, forexample, Schaefer et al., (1989) JACC 14:472-480; Shreve et al., (1986)Magn. Reson. Med. 3:336-340; Wolf, G. L., (1984) Physiol. Chem. Phys.Med. NMR 16:93-95; Wesbey et al., (1984) Physiol. Chem. Phys. Med. NMR16:145-155; Runge et al., (1984) Invest. Radiol. 19:408-415. Examples ofsuitable fluorescent labels include a fluorescein label, anisothiocyalate label, a rhodamine label, a phycoerythrin label, aphycocyanin label, an allophycocyanin label, an ophthaldehyde label, afluorescamine label, etc. Examples of chemiluminescent labels include aluminal label, an isoluminal label, an aromatic acridinium ester label,an imidazole label, an acridinium salt label, an oxalate ester label, aluciferin label, a luciferase label, an aequorin label, etc.

Those of ordinary skill in the art will know of other suitable labelswhich may be employed in accordance with the present invention. Thebinding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known and used by thoseof ordinary skill in the art. Typical techniques are described byKennedy et al., (1976) Clin. Chim. Acta 70, 1-31; and Schurs et al.,(1977) Clin. Chim. Acta 81, 1-40. Coupling techniques mentioned in thelatter are the glutaraldehyde method, the periodate method, thedimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide estermethod. All of these methods are incorporated by reference herein.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

EXAMPLE 1 Amplification of Serine Proteases Using Redundant and SpecificPrimers

Only cDNA preparations deemed free of genomic DNA were used for geneexpression analysis. Redundant primers were prepared for serineproteases, metallo-proteases and cysteine protease. The primers weresynthesized to consensus sequences of amino acid surrounding thecatalytic triad for serine proteases, viz. histidine . . . aspartate . .. and serine. The sequences of both sense (histidine & aspartate) andantisense (aspartate and serine) redundant primers are shown in Table 2.

TABLE 2 SEQ ID PCR Primers 5′→3′ NO: Redundant Primers: Serine Protease(histidine) = S1 tgggtigtiacigcigcica(ct)tg  1 Serine Protease (asparticacid) = AS1 a(ag)ia(ag)igciatitcitticc  2 Serine Protease (serine)= AS11 a(ag)iggiccicci(cg)(ta)(ag)tcicc  3 Cysteine Protease - senseca(ag)ggica(ag)tg(ct)ggi(ta)(cg)itg(ct)tgg  4 Cysteine Protease -antisense taiccicc(ag)tt(ag)caicc(ct)tc  5 Metallo Protease - sensecci(ac)gitg(tc)ggi(ga)(ta)icciga  6 Metallo Protease - antisensett(ag)tgicciai(ct)tc(ag)tg  7 Specific Primers: Serine Protease (SCCE)= sense tgtcccgatggcgagtgttt  8 Serine Protease (SCCE) = antisensecctgttggccatagtactgc  9 Serine Protease (SCCE) = senseagatgaatgagtacaccgtg 10 Serine Protease (SCCE) = antisenseccagtaagtccttgtaaacc 11 Serine Protease (Comp B) = senseaagggacacgagagctgtat 12 Serine Protease (Comp B) = antisenseaagtggtagttggaggaagc 13 Serine Protease (Protease M) = sensectgtgatccaccctgactat 20 Serine Protease (Protease M) = antisensecaggtggatgtatgcacact 21 Serine Protease (TADG12) = sense (Ser10-s)gcgcactgtgtttatgagat 22 Serine Protease (TADG12) = antisense (Ser10-as)ctctttggcttgtacttgct 23 Serine Protease (TADG13) = sensetgagggacatcattatgcac 24 Serine Protease (TADG13) = antisensecaagttttccccataattgg 25 Serine Protease (TADG14) = senseacagtacgcctgggagacca 26 Serine Protease (TADG14) = antisensectgagacggtgcaattctgg 27 Cysteine Protease (Cath-L) = senseattggagagagaaaggctac 14 Cysteine Protease (Cath-L) = antisensecttgggattgtacttacagg 15 Metallo Protease (PUMP1) = sensecttccaaagtggtcacctac 16 Metallo Protease (PUMP1) = antisensectagactgctaccatccgtc 17

EXAMPLE 2 Carcinoma Tissue

Several protease entities were identified and subcloned from PCRamplification of cDNA derived from serous cystadenocarcinomas.Therefore, the proteases described herein are reflective of surfaceactivities for this type of carcinoma, the most common form of ovariancancer. It was also shown that PCR amplification bands unique to themucinous tumor type and the clear cell type have similar base pair size.About 20-25% of ovarian cancers are classified as either mucinous, clearcell, or endometrioid.

EXAMPLE 3 Ligation, Transformation and Sequencing

To determine the identity of the PCR products, all the appropriate bandswere ligated into Promega T-vector plasmid and the ligation product wasused to transform JM109 cells (Promega) grown on selective media. Afterselection and culturing of individual colonies, plasmid DNA was isolatedby means of the WIZARD MINIPREP™ DNA purification system (Promega).Inserts were sequenced using a Prism Ready Reaction Dydeoxy Terminatorscycle sequencing kit (Applied Biosystems). Residual dye terminators wereremoved from the completed sequencing reaction using a CENTRISEP SPIN™column (Princeton Separation), and samples were loaded into an AppliedBiosystems Model 373A DNA sequencing system. The results of subcloningand sequencing for the serine protease primers are summarized in Table3.

TABLE 3 Subclone Primer Set Gene Candidate 1 His-Ser SCCE 2 His-Ser SCCE3 His-Ser Compliment B 4 His-Asp Cofactor 1 5 His-Asp TADG-12* 6 His-SerTADG-13* 7 His-Ser TADG-14* 8 His-Ser Protease M 9 His-Ser TADG-15**indicates novel proteases

EXAMPLE 4 Cloning and Characterization

Cloning and characterization of new gene candidates was undertaken toexpand the panel representative of extracellular proteases specific forovarian carcinoma subtypes. Sequencing of the PCR products derived fromtumor cDNA confirms the potential candidacy of these genes. The threenovel genes all have conserved residues within the catalytic triadsequence consistent with their membership in the serine protease family.

PCR products amplified from normal and carcinoma cDNAs were comparedusing sense-histidine and antisense-aspartate as well as sense-histidineand antisense-serine. The anticipated PCR products of approximately 200bp and 500 bp for those pairs of primers were observed. Aspartate isapproximately 50-70 amino acids downstream from histidine and serine isabout 100-150 amino acids toward the carboxy end from histidine.

FIG. 1 shows a comparison of PCR products derived from normal andcarcinoma cDNA as shown by staining in an agarose gel. Two distinctbands in Lane 2 were present in the primer pair sense-His/antisense ASP(AS1) and multiple bands of about 500 bp are noted in the carcinoma lanefor the sense-His/antisense-Ser (AS2) primer pairs in Lane 4.

EXAMPLE 5 Quantitative PCR

The mRNA overexpression of SCCE was detected and determined usingquantitative PCR. Quantitative PCR was performed generally according tothe method of Noonan et al. (1990). The following oligonucleotideprimers were used: SCCE: forward 5′-AGATGAATGAGTACACCGTG-3′ (SEQ ID NO:10), and reverse 5′-CCAGTAAGTCCTTGTAAACC-3′ (SEQ ID NO: 11); andβ-tubulin: forward 5′-TGCATTGACAACGAGGC-3′ (SEQ ID NO: 18), and reverse5′-CTGTCTTGA CATTGTTG-3′ (SEQ ID NO: 19). β-tubulin was utilized as aninternal control.

The predicted sizes of the amplified genes were 339 bp for SCCE and 454bp for β-tubulin. The primer sequences used in this study were designedaccording to the cDNA sequences described by Hansson et al. (1994) forSCCE, and Hall et al. (1983) for β-tubulin. The PCR reaction mixtureconsisted of cDNA derived from 50 ng of mRNA converted by conventionaltechniques, 5 μmol of sense and antisense primers for both the SCCE geneand the β-tubulin gene, 200 μmol of dNTPs, 5 μCi of α-³²PdCTP and 0.25units of Taq DNA polymerase with reaction buffer (Promega) in a finalvolume of 25 μl. The target sequences were amplified in parallel withthe β-tubulin gene. Thirty cycles of PCR were carried out in a ThermalCycler (Perkin-Elmer Cetus). Each cycle of PCR included 30 sec ofdenaturation at 95° C., 30 sec of annealing at 63° C. and 30 sec ofextension at 72° C. It was previously established and confirmed for SCCEthat co-amplification with β-tubulin under these conditions for 30cycles remain linear for both products.

The PCR products were separated on 2% agarose gels and the radioactivityof each PCR product was determined by using a Phospho Imager (MolecularDynamics). In the present study, expression of SCCE was calculated asthe ratio (SCCE/β-tubulin) as measured by phosphoimager. Theoverexpression cut-off value was defined as the mean value for normalovary +2SD. The student's t test was used for the comparison of the meanvalues of normal ovary and tumors.

Experiments comparing PCR amplification in normal ovary and ovariancarcinoma suggested overexpression and/or alteration in mRNA transcriptin tumor tissues. Northern blot analysis of TADG-14 confirms atranscript size of 1.4 kb and data indicate overexpression in ovariancarcinoma (FIG. 2). Isolation and purification using both PCR and aspecific 250 bp PCR product to screen positive plaques yielded a 1.2 kbclone of TADG-14. Other proteases were amplified by the same methodusing the appropriate primers from Table 2.

EXAMPLE 6 Tissue Bank

A tumor tissue bank of fresh frozen tissue of ovarian carcinomas asshown in Table 4 was used for evaluation. Approximately 100 normalovaries removed for medical reasons other than malignancy were obtainedfrom surgery and were available as controls.

TABLE 4 Total Stage I/11 Stage III/IV No Stage Serous Malignant 166 15140 8 LMP 16 9 7 0 Benign 12 0 0 12 Mucinous Malignant 26 6 14 6 LMP 2825 3 0 Benign 3 0 0 3 Endometrioid Malignant 38 17 21 0 LMP 2 2 0 0Benign 0 0 0 0 Other* Malignant 61 23 29 9 LMP 0 0 0 0 Benign 5 0 0 5*Other category includes the following tumor types: Brenner's tumor,thecoma, teratoma, fibrothecoma, fibroma, granulosa cell, clear cell,germ cell, mixed mullerian, stromal, undifferentiated, and dysgerminoma.

From the tumor bank, approximately 100 carcinomas were evaluatedencompassing most histological sub-types of ovarian carcinoma, includingborderline or low-malignant potential tumors and overt carcinomas. Theapproach included using mRNA prepared from fresh frozen tissue (bothnormal and malignant) to compare expression of genes in normal, lowmalignant potential tumors and overt carcinomas. The cDNA prepared frompolyA+ mRNA was deemed to be genomic DNA free by checking allpreparations with primers that encompassed a known intron-exon splicesite using both β-tubulin and p53 primers.

EXAMPLE 7 Northern Blots Analysis

Significant information can be obtained by examining the expression ofthese candidate genes by Northern blot. Analysis of normal adultmulti-tissue blots offers the opportunity to identify normal tissueswhich may express the protease. Ultimately, if strategies for inhibitionof proteases for therapeutic intervention are to be developed, it isessential to appreciate the expression of these genes in normal tissueif and when it occurs.

Northern panels for examining expression of genes in a multi-tissuenormal adult as well as fetal tissue are commercially available(CLONTECH). Such evaluation tools are not only important to confirm theoverexpression of individual transcripts in tumor versus normal tissues,but also provides the opportunity to confirm transcript size, and todetermine if alternate splicing or other transcript alteration may occurin ovarian carcinoma.

Northern blot analysis was performed as follows: 10 μg of mRNA wasloaded onto a 1% formaldehyde-agarose gel, electrophoresed and blottedonto a HyBond-N⁺™ nylon membrane (Amersham). ³²P-labeled cDNA probeswere made using Prime-a-Gene Labeling System™ (Promega). The PCRproducts amplified by specific primers were used as probes.

Blots were prehybridized for 30 min and then hybridized for 60 min at68° C. with ³²P-labeled cDNA probe in ExpressHyb™ Hybridization Solution(CLONTECH). Control hybridization to determine relative gel loading wasaccomplished using the β-tubulin probe.

Normal human tissues including spleen, thymus, prostate, testis, ovary,small intestine, colon, peripheral blood leukocyte, heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas and normalhuman fetal tissues (Human Multiple Tissue Northern Blot; CLONTECH) wereall examined using the same hybridization procedure.

EXAMPLE 8 PCR Products Corresponding to Serine, Cysteine andMetallo-Proteases

Based on their unique expression in either low malignant potentialtumors or carcinomas, PCR-amplified cDNA products were cloned andsequenced and the appropriate gene identified based upon nucleotide andamino acid sequences stored in the GCG and EST databases. FIGS. 3, 4 & 5show the PCR product displays comparing normal and carcinomatous tissuesusing redundant primers for serine proteases (FIG. 3), for cysteineproteases (FIG. 4) and for metallo-proteases (FIG. 5). Note thedifferential expression in the carcinoma tissues versus the normaltissues. The proteases were identified using redundant cDNA primers(Table 2) directed towards conserved sequences that are associated withintrinsic enzyme activity, for serine proteases, cysteine proteases andmetallo-proteases, by comparing mRNA expression in normal, low malignantpotential and overt ovarian carcinoma tissues according to Sakanari etal. (1989).

EXAMPLE 9 Serine Proteases

For the serine protease group, using the histidine domain primer sense,S1, in combination with antisense primer AS2, the following proteaseswere identified:

(a) Hepsin, a trypsin-like serine protease cloned from hepatoma cellsshown to be a cell surface protease essential for the growth of hepatomacells in culture and highly expressed in hepatoma tumor cells (FIG. 3,Lane 4);

(b) Complement factor B protease (human factor IX), a protease involvedin the coagulation cascade and associated with the production andaccumulation of fibrin split products associated with tumor cells (FIG.3, Lane 4). Compliment factor B belongs in the family of coagulationfactors X (Christmas factor). As part of the intrinsic pathway,compliment factor B catalyzes the proteolytic activation of coagulationfactor X in the presence of Ca²⁺ phospholipid and factor VIIIa e5; and

(c) A stratum corneum chymotryptic enzyme (SCCE) serine proteaseinvolved in desquarnation of skin cells from the human stratum corneum(FIG. 3, Lane 4). SCCE is expressed in keratinocytes of the epidermisand functions to degrade the cohesive structures in the cornified layerto allow continuous skin surface shedding.

EXAMPLE 10 Cysteine Proteases

In the cysteine protease group, using redundant sense and anti-senseprimers for cysteine proteases, one unique PCR product was identified byoverexpression in ovarian carcinoma when compared to normal ovariantissue (FIG. 4, Lanes 3-5). Cloning and sequencing this PCR productidentified a sequence of Cathepsin L which is a lysomal cysteineprotease whose expression and secretion is induced by malignanttransformation, growth factors and tumor promoters. Many human tumors(including ovarian) express high levels of Cathepsin L. Cathepsin Lcysteine protease belongs in the stromolysin family and has potentelastase and collagenase activities. Published data indicates increasedlevels in the serum of patients with mucinous cystadenocarcinoma of theovary. It has not heretofore been shown to be expressed in other ovariantumors.

EXAMPLE 11 Metallo-Proteases

Using redundant sense and anti-sense primers for the metallo-proteasegroup, one unique PCR product was detected in the tumor tissue which wasabsent in normal ovarian tissue (FIG. 5, Lanes 2-5). Subcloning andsequencing this product indicates it has complete homology in theappropriate region with the so-called PUMP-1 (MMP-7) gene. Thiszinc-binding metallo-protease is expressed as a proenzyme with a signalsequence and is active in gelatin and collagenase digestion. PUMP-1 hasalso been shown to be induced and overexpressed in 9 of 10 colorectalcarcinomas compared to normal colon tissue, suggesting a role for thissubstrate in the progression of this disease.

EXAMPLE 12 mRNA Expression of SCCE in Ovarian Tumors

To evaluate mRNA expression of SCCE in ovarian tumors, semi-quantitativePCR was performed. A preliminary study confirmed the linearity of thePCR amplification according to the methods of Shigemasa et al. (1997)and Hall et al. (1983). FIG. 6 shows an example of comparative PCR usingSCCE primers co-amplified with the internal control β-tubulin primers.Analysis of the data as measured using the phosphoimager and compared asratios of expression (SCCE/β-tubulin) indicate that SCCE expression iselevated in tumor cases 19, 14, 15, 16, 43, 23, 36 and 37 compared tothat of normal ovaries.

To confirm the results of the initial quantitative PCR and to examinethe size of the transcript, Northern blot hybridization was performed inrepresentative cases of each histological type of carcinoma (FIG. 7A).Northern blot hybridization with a ³²P-labeled SCCE probe (nucleotides232-570) revealed 1.2 kb and 2.0 kb transcripts, as reported previouslyin normal skin tissue (Hansson et al., 1994). Those tumor cases whichshowed overexpression of SCCE by quantitative PCR also showed intensebands of SCCE transcript expression by Northern blot analysis includingserous, mucinous, endometrioid and clear cell carcinoma. No transcriptswere detected in normal ovarian tissue (Lane 1). Normal human tissues(spleen, thymus, prostate, testis, ovary, small intestine, colon,peripheral blood leukocyte, heart, brain, placenta, lung, liver,skeletal muscle, kidney and pancreas) and normal human fetal tissues(brain, lung, liver and kidney) examined by Northern blot analysisshowed no visible SCCE transcripts (FIGS. 7B & 7C). Blots for normalhuman adult tissues and fetal tissues were subsequently probed toconfirm the presence of β-tubulin transcripts.

Table 5 summarizes the results of the evaluation of SCCE expression in10 individual normal ovarian tissues and 44 ovarian carcinomas. Overall,SCCE mRNA overexpression (overexpression=mean value for normalovary+2SD) was found in 8 of 12 LMP tumors (66.7%) and 25 of 32carcinoma cases (78.1%) with p values of <0.05 and <0.001 respectively(FIG. 8). Overexpression of SCCE transcripts was detected in all ovariancarcinoma subtypes and in both early stage and late stage tumor samples.In the five cases where positive confirmation of lymph node metastasiswas identified, all five cases showed overexpression of SCCE at a levelof more than four standard deviations above the level for normal ovary.It should be noted that three of these tumors were classified as lowmalignant potential tumors (all serous adenomas) suggesting a possiblerelationship between the progression of early stage disease to the lymphwhen overexpression of SCCE is manifest.

The expression ratio (mean value±SD) for normal ovary was determined as0.046±0.023, for LMP tumors as 0.405±0.468 and for carcinoma as0.532±0.824 (Table 6). From a histological point of view, overexpressionof SCCE was observed in 23 of 26 serous tumors (88.5%) including 6 of 7LMP tumors and 17 of 19 carcinomas. However only 4 of 12 mucinous tumors(33.3%) including 2 of 5 LMP tumors and 2 of 7 carcinomas showedoverexpression of SCCE. For endometrioid and clear cell carcinoma,stratum corneum chymotryptic enzyme was found to be overexpressed in all6 cases (Table 6).

TABLE 5 Patient Characteristics and Expression of SCCE Gene mRNAexpression^(c) Case Histological Type^(a) Stage/Grade LN^(b) SCCE 1normal ovary n 2 normal ovary n 3 normal ovary n 4 normal ovary n 5normal ovary n 6 normal ovary n 7 normal ovary n 8 normal ovary n 9normal ovary n 10 normal ovary n 11 s adenoma (LMP) 1/1 n 4+ 12 sadenoma (LMP) 1/1 NE n 13 s adenoma (LMP) 1/1 NE 2+ 14 s adenoma (LMP)1/1 n 4+ 15 s adenoma (LMP) 3/1 p 4+ 16 s adenoma (LMP) 3/1 p 4+ 17 sadenoma (LMP) 3/1 p 4+ 18 m adenoma (LMP) 1/1 NE 4+ 19 m adenoma (LMP)1/1 n 4+ 20 m adenoma (LMP) 1/1 n n 21 m adenoma (LMP) 1/1 NE n 22 madenoma (LMP) 1/1 NE n 23 s carcinoma 1/2 n 4+ 24 s carcinoma 1/3 n 4+25 s carcinoma 3/1 NE 4+ 26 s carcinoma 3/2 NE 4+ 27 s carcinoma 3/2 p4+ 28 s carcinoma 3/2 NE 4+ 29 s carcinoma 3/3 NE 4+ 30 s carcinoma 3/3NE 4+ 31 s carcinoma 3/3 NE 4+ 32 s carcinoma 3/3 NE 4+ 33 s carcinoma3/3 n 4+ 34 s carcinoma 3/3 NE n 35 s carcinoma 3/3 NE 4+ 36 s carcinoma3/3 NE 4+ 37 s carcinoma 3/3 NE 4+ 38 s carcinoma 3/3 n 4+ 39 scarcinoma 3/2 NE 4+ 40 s carcinoma 3/3 NE 4+ 41 s carcinoma 3/2 NE n 42m carcinoma 1/2 n n 43 m carcinoma 2/2 NE 4+ 44 m carcinoma 2/2 n n 45 mcarcinoma 3/1 NE n 46 m carcinoma 3/2 NE n 47 m carcinoma 3/2 NE n 48 mcarcinoma 3/3 NE 4+ 49 e carcinoma 2/3 n 4+ 50 e carcinoma 3/2 NE 4+ 51e carcinoma 3/3 NE 4+ 52 c carcinoma 1/3 n 4+ 53 c carcinoma 1/1 n 4+ 54c carcinoma 3/2 p 4+ ^(a)s; serous, m; mucinous, e; endometrioid, c;clear cell; ^(b)LN; lymph node metastasis, p; positive, n; negative, NE;not examined; ^(c)n, normal range is equal to Mean ± 2SD, 2+; Mean + 2SDto + 4SD, 4+; Mean + 4SD or greater

TABLE 6 Overexpression of SCCE in Ovarian Carcinoma Overexpression N ofSCCE Expression Ratio^(a) Normal 10  0 (0%) 0.046 ± 0.023 LMP 12  8(66.7%) 0.045 ± 0.468 serous 7  6 (85.7%) 0.615 ± 0.518 mucinous 5  2(40.0%) 0.111 ± 0.117 Carcinomas 32 25 (78.1%) 0.532 ± 0.824 serous 1917 (89.9%) 0.686 ± 1.027 mucinous 7  2 (28.6%) 0.132 ± 0.265 endometriod 3 3 (100%) 0.511 ± 0.205 clear cell 3  3 (100%) 0.515 ± 0.007 ^(a)Theratio of expression level of SCCE to β-tubulin

EXAMPLE 13 Western Blot Analysis

Polyclonal rabbit antibodies were generated by immunization with acombination of 2 poly-lysine linked multiple Ag peptides derived fromSCCE protein sequences PLQILLLSLALE (SEQ ID NO: 28) and SFRHPGYSTQTH(SEQ ID NO: 29). Approximately 20 ng of MDA-MBA-435S and HeLa celllysates were separated on a 15% SDS-PAGE gel and electroblotted to PVDFat 100 V for 40 minutes at 4° C. The proteins were fixed to the membraneby incubation in 50% MeOH for 10 minutes. The membrane was blockedovernight in TBS, pH 7.8 containing 0.2% non-fat milk. Primary antibodywas added to the membrane at a dilution of 1:100 in 0.2% milk/TBS andincubated for 2 hours at room temperature. The blot was washed andincubated with a 1:3000 dilution of alkaline-phosphatase conjugated goatanti-rabbit IgG (BioRad) for one hour at room temperature. The blot waswashed and incubated with a chemiluminescent substrate before a 10second exposure to X-ray film for visualization.

Two cell lines HeLa and MDA-MB-435S previously shown to express mRNAtranscripts were examined by Western blot to confirm the presence ofSCCE protein. FIG. 9 indicates that polyclonal antibodies developed topeptides (12 mers bound to polylysine) derived from the amino andcarboxy termini of SCCE bind a protein of approximately 30 kDa incytosolic extracts of HeLa and MDA-MB435S cells. The ovarian tumor cellline CAOV3 was also examined for SCCE expression and a protein productcould not be detected (data not shown). This molecular size proteinagrees with the anticipated and known parameters for the SCCE protein.It should be noted that only a single band was detected by Western blotanalysis of cytosolic protein. It might be anticipated that the SCCEprotein prior to secretion would be present in the inactivated parentform i.e. the seven amino terminal peptide removed on activation wouldstill be present on the enzyme. In this pre-active form of the enzyme itwould be anticipated that the apparent molecular weight on Western blotwould be about 30 kDa.

EXAMPLE 14 Immunohistochemistry

Immunohistochemical localization of SCCE antigen was examined usingnormal ovaries, mucinous LMP tumor and adenocarcinomas (including serousadenocarcinomas, mucinous adenocarcinoma and clear cell carcinomas) inthe same series of the samples for mRNA isolation. Formalin fixed andparaffin embedded sections, 4 μm thick, were cut and mounted onaminopropyltriethoxysilane treated slides. Slides were routinelydeparaffinized with xylene and rehydrated with a series of ethanolwashes. Nonenzymatic antigen retrieval was performed by processing usingmicrowave heat treatment in 0.01 M sodium citrate buffer (pH 6.0).Immunohistochemical staining was performed manually using theavidin-biotin peroxidase complex technique (Vectastain Elite ABC kit,Vector Laboratories). Anti-SCCE rabbit polyclonal antibody was generatedby immunization with a combination of 2 poly-lysine linked multiple Agpeptide derived from the SCCE protein-sequences.

This indirect immunoperoxidase staining procedure was performed at roomtemperature. Endogenous peroxidase and nonspecific background stainingwere blocked by incubating slides with methanol with 0.3% H₂O₂ for 30minutes. After washing with phosphate-buffered saline (PBS) for 10minutes, sections were incubated with biotinylated anti-rabbit IgG for30 minutes. After washing with PBS for 10 minutes, slides were incubatedwith ABC reagent for 30 minutes. The final products were visualized byusing AEC substrate system (DAKO Corporation) and sections werecounterstained with Mayer hematoxylin for 20 seconds before mounting.Positive controls and negative controls were used for each section.Negative controls were performed by using normal rabbit serum instead ofthe primary antibody. All experiments were duplicated. The stainedslides were examined microscopically by 3 observers. More than 10% ofpositive tumor cells was the criterion for a 1+ positive staining andmore than 50% of positive tumor cells was the criterion for a 2+positive staining.

To further confirm the presence of the SCCE protein in ovarian tumorcells as opposed to its elaboration by supporting stromal or bloodvessel cells, both normal ovarian epithelia and ovarian tumor tissuewere examined by immunohistochemistry using the polyclonal antiserumdescribed above. All 14 ovarian tumors showed positive staining of SCCE,whereas normal ovarian surface epithelium showed very weak expression ofSCCE antigen (FIG. 10A). 8 of 10 serous adenocarcinomas, 1 of 1 mucinousadenocarcinoma, and 2 of 2 clear cell carcinomas showed 2+ positivestaining (more than 50% of positive tumor cells) of SCCE (Table 7).FIGS. 10C and 10E show that stratum corneum chymotryptic enzyme stainingis localized to the cytoplasm and the cell membrane of ovarian tumorcells. The negative control of each case was also performed, wherein theresult showed no nonspecific staining of stratum corneum chymotrypticenzyme (FIGS. 10B, 10D and 10F). Staining of normal ovarian epithelialcells showed little SCCE expression (FIG. 10A).

TABLE 7 Immunohistochemical Expression of SCCE Protein in Normal Ovaryand Ovarian Tumor Lab NO: Histology SCCE normal ovary weak + normalovary weak + normal ovary weak + normal ovary weak + normal ovary weak +normal ovary weak + 1036 mucinous LMP + 475 serous carcinoma + 465serous carcinoma ++ 464 serous carcinoma ++ 1039 serous carcinoma ++ 960serous carcinoma ++ 962 serous carcinoma ++ 1551 serous carcinoma ++1813 serous carcinoma ++ 1817 serous carcinoma + 1819 serous carcinoma++ 1244 mucinous carcinoma ++ 947 clear cell carcinoma ++ 948 clear cellcarcinoma ++

EXAMPLE 15 Summary of Proteases Detected Herein

Most of the above-listed proteases were identified from thesense-His/antisense-Ser primer pair, yielding a 500 bp PCR product (FIG.1, Lane 4). Some of the enzymes are familiar, a short summary of eachfollows.

Hepsin

Hepsin is a trypsin-like serine protease cloned from hepatoma cells.Hepsin is an extracellular protease (the enzyme includes a secretionsignal sequence) which is anchored in the plasma membrane by its aminoterminal domain, thereby exposing its catalytic domain to theextracellular matrix. Hepsin has also been shown to be expressed inbreast cancer cell lines and peripheral nerve cells. Hepsin has neverbefore been associated with ovarian carcinoma. Specific primers for thehepsin gene were synthesized and the expression of hepsin examined usingNorthern blots of fetal tissue and ovarian tissue (both normal andovarian carcinoma).

FIG. 11A shows that hepsin was expressed in ovarian carcinomas ofdifferent histologic types, but not in normal ovary. FIG. 11B shows thathepsin was expressed in fetal liver and fetal kidney as anticipated, butat very low levels or not at all in fetal brain and lung. FIG. 11C showsthat hepsin overexpression is not observed in normal adult tissue.Slight expression above the background level is observed in the adultprostate. The mRNA identified in both Northern blots was the appropriatesize for the hepsin transcript.

The expression of hepsin was examined in 10 normal ovaries and 44ovarian tumors using specific primers to β-tubulin and hepsin in aquantitative PCR assay. Expression is presented as the ratio of³²P-hepsin band to the internal control, the ³²P-β-3-tubulin band.Hepsin mRNA is highly overexpressed in most histopathologic types ofovarian carcinomas including some low malignant potential tumors (seeFIGS. 12A & 12B). Most noticeably, hepsin is highly expressed in serous,endometrioid and clear cell tumors tested. It is highly expressed insome mucinous tumors, but it is not overexpressed in the majority ofsuch tumors.

Stratum Corneum Chymotrypsin Enzyme (SCCE)

The PCR product identified was the catalytic domain of thesense-His/antisense-Ser of the SCCE enzyme. This extracellular proteasewas cloned, sequenced and shown to be expressed on the surface ofkeratinocytes in the epidermis. SCCE is a chymotrypsin-like serineprotease whose function is suggested to be in the catalytic degradationof intercellular cohesive structures in the stratum corneum layer of theskin. This degradation allows continuous shedding (desquamation) ofcells from the skin surface. The subcellular localization of SCCE is inthe upper granular layer in the stratum corneum of normalnon-palmoplantar skin and in the cohesive parts of hypertrophic plantarstratum corneum. SCCE is exclusively associated with the stratum corneumand has not been shown to be expressed in any carcinomatous tissues.

Northern blots were probed with the PCR product to determine expressionof SCCE in fetal tissue and ovarian carcinoma (FIGS. 7A, 7B and 7C).Noticeably, detection of SCCE messenger RNA on the fetal Northern wasalmost non-existent (a problem with the probe or the blot was excludedby performing the proper controls). A faint band appeared in fetalkidney. On the other hand, SCCE mRNA is abundant in the ovariancarcinoma mRNA (FIG. 7A). Two transcripts of the correct size areobserved for SCCE. The same panel of cDNA used for SCCE analysis wasused for SCCE expression.

No SCCE expression was detected in the normal ovary lane of the Northernblot. A comparison of all candidate genes, including a loading marker(β-tubulin), was shown to confirm that this observation was not a resultof a loading bias. Quantitative PCR using SCCE primers, along withβ-tubulin internal control primers, confirmed the overexpression of SCCEmRNA in carcinoma of the ovary with no expression in normal ovariantissue (FIG. 6). FIG. 8 shows the ratio of SCCE to the β-tubulininternal standard in 10 normal and 44 ovarian carcinoma tissues. Again,it is observed that SCCE is highly overexpressed in ovarian carcinomacells. It is also noted that some mucinous tumors overexpress SCCE, butthe majority do not.

Protease M

Protease M was identified from subclones of the His-ser primer pair.This protease was cloned by Anisowicz, et al., and shown to beoverexpressed in carcinomas. A evaluation indicates that this enzyme isoverexpressed in ovarian carcinoma (FIG. 13).

Cofactor I and Complement Factor B

Several serine proteases associated with the coagulation pathway werealso subcloned. Examination of normal and ovarian carcinomas byquantitative PCR for expression of these enzymes, it was noticeable thatthis mRNA was not clearly overexpressed in ovarian carcinomas whencompared to normal ovarian tissue. It should be noted that the samepanel of tumors was used for the evaluation of each candidate protease.

EXAMPLE 16 Summary of Previously Unknown Proteases Detected HereinTADG-12

TADG-12 was identified from the primer pairs, sense-His/antisense-Asp(see FIG. 1, Lanes 1 & 2). Upon subcloning both PCR products in lane 2,the 200 bp product had a unique protease-like sequence not included inGenBank. This 200 bp product contains many of the conserved amino acidscommon for the His-Asp domain of the family of serine proteins. Thesecond and larger PCR product (300 bp) was shown to have a high degreeof homology with TADG-12 (His-Asp sequence), but also containedapproximately 100 bp of unique sequence. Synthesis of specific primersand the sequencing of the subsequent PCR products from three differenttumors demonstrated that the larger PCR product (present in about 50% ofovarian carcinomas) includes an insert of about 100 bp near the 5′ end(and near the histidine) of the sequence. This insert may be a retainedgenomic intron because of the appropriate position of splice sites andthe fact that the insert does not contain an open reading frame (seeFIG. 14). This suggests the possibility of a splice site mutation whichgives rise to retention of the intron, or a translocation of a sequenceinto the TADG-12 gene in as many as half of all ovarian carcinomas.

TADG-13 and TADG-14

Specific primers were synthesized for TADG-13 and TADG-14 to evaluateexpression of genes in normal and ovarian carcinoma tissue. Northernblot analysis of ovarian tissues indicates the transcript for theTADG-14 gene is approximately 1.4 kb and is expressed in ovariancarcinoma tissues (FIG. 15A) with no noticeable transcript presence innormal tissue. In quantitative PCR studies using specific primers,increased expression of TADG-14 in ovarian carcinoma tissues was notedcompared to a normal ovary (FIG. 15B). The presence of a specific PCRproduct for TADG-14 in both an HeLa library and an ovarian carcinomalibrary was also confirmed. Several candidate sequences corresponding toTADG-14 have been screened and isolated from the HeLa library.

Clearly from sequence homology, these genes fit into the family ofserine proteases. TADG-13 and TADG-14 are, however, heretoforeundocumented genes which the specific primers of the invention allow tobe evaluated in normal and tumor cells, and with which the presence orabsence of expression of these genes is useful in the diagnosis ortreatment selection for specific tumor types.

PUMP-1

In a similar strategy using redundant primers to metal binding domainsand conserved histidine domains, a differentially expressed PCR productidentical to matrix metallo-protease 7 (MMP-7) was identified, hereincalled PUMP-1. Using specific primers for PUMP-1, PCR produced a 250 bpproduct for Northern blot analysis.

MMP-7 or PUMP-1 is differentially expressed in fetal lung and kidneytissues. FIG. 16A compares PUMP-1 expression in normal ovary andcarcinoma subtypes using Northern blot analysis. Notably, PUMP-1 isexpressed in ovarian carcinoma tissues, and again, the presence of atranscript in normal tissue was not detected. FIG. 16B shows theexpression of PUMP-1 in human fetal tissue, while no transcript could bedetected in either fetal brain or fetal liver. FIG. 16C shows thatPUMP-1 overexpression is not observed in normal adult tissue.Quantitative PCR comparing normal versus ovarian carcinoma expression ofthe PUMP-1 mRNA indicates that this gene is highly expressed in serouscarcinomas, including most low malignant serous tumors, and is, again,expressed to a lesser extent in mucinous tumors (FIGS. 17A & 17B).PUMP-1, however, is so far the protease most frequently foundoverexpressed in mucinous tumors (See Table 8 below).

Cathepsin-L

Using redundant cysteine protease primers to conserved domainssurrounding individual cysteine and histidine residues, the cathepsin-Lprotease was identified in several serous carcinomas. An initialexamination of the expression of cathepsin L in normal and ovarian tumortissue indicates that transcripts for the cathepsin-L protease arepresent in both normal and tumor tissues (FIG. 18). However, itspresence or absence in combination with other proteases of the presentinvention permits identification of specific tumor types and treatmentchoices.

CONCLUSION

Redundant primers to conserved domains of serine, metallo-, and cysteineproteases have yielded a set of genes whose mRNAs are overexpressed inovarian carcinoma.

The genes which are clearly overexpressed include the serine proteaseshepsin, SCCE, protease M, TADG12, TADG14 and the metallo-protease PUMP-1(see FIG. 19 and Table 8). Northern blot analysis of normal and ovariancarcinoma tissues indicated overexpression of hepsin, SCCE, PUMP-1 andTADG-14. A β-tubulin probe to control for loading levels was included.

TABLE 8 Overexpression of Proteases in Ovarian Tumors Type N Hepsin SCCEPump-1 Protease M Normal 10 0% (0/10)   0% (0/10)   0% (0/10)   0%(0/10) LMP 12 58.3% (7/12) 66.7% (8/12) 75.0% (9/12)   75% (9/12) serous7 85.7% (6/7) 85.7% (6/7) 85.7% (6/7)  100% (7/7) mucinous 5 20.0% (1/5)40.0% (2/5)   60% (3/5) 40.0% (2/5) Carcinoma 32 84.4% (27/32) 78.1%(25/32) 81.3% (26/32) 90.6% (29/32) serous 19 94.7% (18/19) 89.5%(17/19) 78.9% (15/19) 94.7% (18/19) mucinous 7 42.9% (3/7) 28.6% (2/7)71.4% (5/7) 85.7% (6/7) endometr. 3  100% (3/3)  100% (3/3)  100% (3/3) 100% (3/3) clear cell 3  100% (3/3)  100% (3/3)  100% (3/3) 67.7% (2/3)

Discussion

For the most part, these proteins previously have not been associatedwith the extracellular matrix of ovarian carcinoma cells. No panel ofproteases which might contribute to the growth, shedding, invasion andcolony development of metastatic carcinoma has been previouslydescribed, including the three new candidate serine proteases which areherein disclosed. The establishment of an extracellular protease panelassociated with either malignant growth or malignant potential offersthe opportunity for the identification of diagnostic or prognosticmarkers and for therapeutic intervention through inhibition or downregulation of these proteases.

The availability of the instant gene-specific primers coding for theappropriate region of tumor specific proteases allows for theamplification of a specific cDNA probe using Northern and Southernanalysis, and their use as markers to detect the presence of the cancerin tissue. The probes also allow more extensive evaluation of theexpression of the gene in normal ovary versus low malignant potentialtumor, as well as both high- and low-stage carcinomas. The evaluation ofa panel of fresh frozen tissue from all the carcinoma subtypes (Table 4)allowed the determination of whether a protease is expressedpredominantly in early stage disease or within specific carcinomasubtypes. It was also determined whether each gene's expression isconfined to a particular stage in tumor progression and/or is associatedwith metastatic lesions. Detection of specific combinations of proteasesis an identifying characteristic of the specific tumor types and yieldsvaluable information for diagnoses and treatment selection. Particulartumor types may be more accurately diagnosed by the characteristicexpression pattern of each specific tumor.

Specifically, the present invention utilizes primers to the conservedcatalytic triad domain of the serine protease family (viz. His-Asp-Ser).Using such a strategy to display serine protease transcripts found inabundance in carcinoma tissues, with little or no expression in normalovary, SCCE gene was detected.

The overall expectation of the search was to identify cell surface orsecreted products which may promote either tumor growth or metastasis.Confirmation of the presence of SCCE (a secreted serine protease) inovarian tumors was indicated initially by subcloning and sequencing PCRproducts derived from amplification of tumor cDNA using redundant primesto the histidine (sense) and the serine (antisense) conserved domains ofthe serine protease catalytic sequences. Characterization of the SCCEprotease (Egelrud, 1993) indicated that the cohesion between individualcorneocytes in the stratum comeurn, the primary substrate for cellulardesquamation or shedding of skin cells may be degraded by SCCE.Proteolysis of these intercellular matrices is one of the major eventspreceding desquamation. SCCE has only been identified in the stratumcomeurn (Egelrud, 1993; Hansson et al., 1994) and immunohistochemicalstudies confirmed its unique tissue specific expression by theepithelial cells of the stratum comeurn (SondeIl et al., 1994). It wastherefore surprising to discover that this highly conserved expressionof SCCE to skin is obviated when transformation and carcinogenesis ofovarian epithelial cells occurs. The clearly distinctive pattern ofexpression in both low malignant potential tumors and overt carcinomasof the ovary over normal ovarian tissue suggests that the SCCE proteasemay also play a role in shedding or desquamation of ovarian tumor cells.This association is especially well preserved in serous adenocarcinomaswhere disease progression is characterized by early foci of peritonealmetastasis and which may be the result of an early overexpression ofenzymes such as SCCE and consequent tumor cell shedding. Because SCCEand other proteases (e.g. hepsin) are overexpressed in ovarian tumors(again with particularly high overexpression in serous tumors), it seemslikely that a concert of lytic activity at the cell surface may beinvolved in malignant potential. Several aspects of the tumorigenicprocess can be dissected and identified as component parts of such asurface protease concert viz 1) initial expansion of newly transformedcells into the surrounding matrix of supporting tissue of the primaryorgan; 2) desquamation or shedding of tumor cells into the surroundingenvironment; 3) invasion of basement membrane of the target organ ofmetastasis; and 4) activation of mitogenic and angiogenic factors tosupport the newly established metastatic colony.

While it is not yet clear which proteases are the primary agents in eachof these malignant progression steps, the data here indicate thepotential for the involvement of SCCE in the shedding or desquamationphase of this progression. Certain other factors remain to be resolvedeven with regard to SCCE involvement in tumor cell shedding whichinclude activation of SCCE by proteolysis or cleaving of theaminoterminal peptide of the pro-protease. Furthermore, anantileukoprotease which specifically inhibits SCCE activity has beenrecently identified (Wiedow, O. (1995) Isolierung und Charakterisierungvon Serinprotease Inhibitoren der menschlichen Epidermis, Köster,Berlin). The presence of such an inhibitor might effectively inhibitshedding or desquamation of tumor cells as it has been shown to inhibitthe detachment of corneocytes of keratinized skin tissue.

While there remains an intricate interaction between surface proteaseexpression/activation and/or inhibition, it appears likely that aconcert of enzymes which contribute to tumor growth and spread provide amechanism for such a progression. SCCE expression on ovarian tumor cellsurfaces can provide one mechanism by which tumor cells may be shedearly in the tumor progression process of serous carcinomas.

The unique presence of this protease to keratinized stratum corneum andthe present data showing lack of transcript presence in all normal adultand fetal tissues examined support the potential of this secretedextracellular enzyme as a useful marker for ovarian carcinoma. The factthat inhibition of such an activity prevents normal desquamation of skincells also points to the potential of SCCE as a target for inhibition ordown regulation in therapeutic intervention in the spread or metastasisof ovarian carcinoma.

EXAMPLE 17 SCCE Peptides as Target Epitopes for Human CD8⁺ Cytotoxic TCells

Two computer programs were used to identify 9-mer peptides containingbinding motifs for HLA class I molecules. The first, based on a schemedevised by Parker et al (1994), was developed by the Bioinformatics andMolecular Analysis Section (BIMAS) of the Center for InformationTechnology, NIH, and the second, known as SYFPEITHI, was formulated byRammensee and colleagues at the University of Tubingen, Germany.

Peptides that possessed HLA A2.1 binding motifs were synthesized andtested directly for their ability to bind HLA A2.1. This techniqueemploys T2 cells which are peptide transporter-deficient and thusexpress low endogenous HLA class I levels due to inability to loadpeptide and stabilize HLA class I folding for surface expression. It hasbeen showed that addition of exogenous peptides capable of binding HLAA2.1 (A*0201) could increase the number of properly folded HLA A2.1molecules on the cell surface, as revealed by flow cytometry (Nijman etal, 1993).

Monocyte-derived DC were generated from peripheral blood drawn fromnormal adult donors of the appropriate HLA type. Adherent monocytes werecultured in AIM-V (Gibco-BRL) supplemented with GM-CSF and IL-4according to standard techniques (Santin et al, 2000). After 5-6 days,DC maturation was induced by addition of PGE₂, IL-1b and TNFa for afurther 48 h.

Mature DC were loaded with peptide (2×10⁶ DC with 50 mg/ml peptide in 1ml serum-free AIM-V medium for 2 h at 37° C.) and washed once prior toculture with 1×10⁶/ml peripheral blood mononuclear cells (PBMC) in AIM-Vor AIM-V plus 5% human AB serum. The PBMC:DC ratio was between 20:1 and30:1. After 7 days, responder T cells were restimulated withpeptide-loaded, irradiated autologous DC or PBMC at responder:stimulatorratios between 10:1 and 20:1 or 1:1 and 1:10 respectively. At thispoint, cultures were supplemented with recombinant human IL-2 (10-100U/ml), and fed with 50-75% changes of fresh medium plus IL-2 every 24days. T cell lines were established and maintained by peptiderestimulation every 14-21 days. Responder CD8⁺ T cells were purified bypositive selection with anti-CD8-coupled magnetic beads (Dynal, Inc.)after the 2^(nd) or 3^(rd) antigen stimulation.

Peptide-specific cytotoxicity was tested in standard 5-6 h microwell⁵¹Cr-release assays (Nazaruk et al, 1998). Autologous EBV-transformedlymphoblastoid cell lines (LCL) were loaded with peptide (50 mg/ml, 1 hat 37° C.) and subsequently ⁵¹Cr-labeled (50 mCi in 200-300 ml, 1 h at37° C.). Peptide-loaded ⁵¹Cr-labeled LCL were incubated with CD8⁺ Tcells at effector-target ration between 5:1 and 1.25:1. Cytotoxicity wasrecorded as percentage ⁵¹Cr released into culture supernatants.

SCCE Peptide 5-13

SCCE peptide 5-13 (SEQ ID NO: 33) is an HLA A2.1-binding peptide, asrevealed by upregulation of A2.1 expression in T2 cells (data notshown). CD8⁺ CTL specific for SCCE 5-13 killed peptide-loaded autologousLCL, but did not kill control, peptide-free LCL. Heterologous HLAA2.1-expressing peptide-loaded LCL were efficiently killed, but targetslacking HLA A2.1 weren't killed (FIG. 20).

SCCE Peptide 123-131

SCCE peptide 123-131 (SEQ ID NO: 32) is also an HLA A2.1-bindingpeptide, as revealed by upregulation of A2.1 expression in T2 cells(data not shown). CD8⁺ CTL specific for SCCE 123-131 killedpeptide-loaded autologous LCL, but did not kill control, peptide-freeLCL. Heterologous HLA A2.1-expressing peptide-loaded LCL wereefficiently killed, but targets lacking HLA A2.1 were not killed (FIG.21). Natural killer-sensitive K562 cells were not lysed. Cytotoxicityagainst SCCE 123-131 loaded LCL could be blocked with MAb specific for anon-polymorphic HLA class I determinant, confirming that lysis was HLAclass I-restricted.

EXAMPLE 18 CD8⁺ CTL Specific for SCCE Peptide 123-131 RecognizeEndogenously Expressed SCCE Tumor Antigen

To determine whether peptide-specific CD8⁺ CTL are capable ofrecognizing targets that process and present endogenously expressed SCCEtumor antigens, recombinant adenoviruses expressing hepsin and SCCE,both in conjunction with green fluorescent protein (GFP) as a means ofdirectly monitoring expression levels by flow cytometric techniques wereconstructed. It was found that CD8⁺ CTL specific for SCCE 123-131recognize and kill autologous targets infected with recombinantadenoviruses expressing the full-length SCCE antigen (Ad-GFP/SCCE) butdid not recognize targets infected with Ad-GFP/hepsin (FIG. 22). Theseresults show that the SCCE 123-131 peptide is a naturally processed andpresented CTL epitope for SCCE-specific CD8⁺ CTL.

CD8⁺ CTL specific for SCCE 123-131 were tested for their ability to lyseHLA-A*0201-matched, SCCE-expressing CaOV-3 ovarian tumor cells.Peptide-specific CTL efficiently lysed peptide-pulsed LCL. The CTL alsolysed CaOV-3 tumor cells (FIG. 23). Thus, SCCE 123-131 is a CTL epitopethat is processed and presented by ovarian tumor cells. Tumor cell lysiswas partially blocked by addition of mAb specific for HLA class I(W6/32) and HLA-A*0201 (BB7.2), indicating that tumor lysis was HLAclass I and HLA-A*0201-restricted. Control, unpulsed LCL andNK-sensitive K562 cells were not lysed. These results indicate thatovarian tumor recognition and lysis was an antigen-specific event.

The following references were cited herein:

-   Egelrud, J Invest Dermatol 101:200-204 (1993).-   Hall et al., Mol. Cell. Biol., 3: 854-862 (1983).-   Hansson et al., J. Biol. Chem., 269:19420-19426 (1994).-   Nazaruk et al., Blood 91:3875-3883 (1998).-   Nijman et al., Eur. J. Immunol. 23:1215-1219 (1993).-   Noonan et al., Proc. Natl. Acad. Sci. USA, 87:7160-7164 (1990).-   Parker et al., J. Immunol. 152:163-175 (1994).-   Powell et al., Cancer Research, 53:417-422 (1993).-   Sakanari et al., Biochemistry 86:4863-4867 (1989).-   Santin et al., Obstetrics & Gynecology 96:422-430 (2000).-   Santin et al., Am. J. Obstet. Gynecol. 183:601-609 (2000).-   Shigemasa et al., J Soc Gynecol Invest 4:95-102, (1997).-   Sondell et al., J Histochem Cytochem 42:459-465 (1994).-   Torres-Rosedo et al., Proc. Natl. Acad. Sci. USA. 90:7181-7185    (1993).

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

1. A method of monitoring the efficacy of vaccinating an individual withstratum corneum chymotrytic enzyme (SCCE) or SCCE peptide, said methodcomprises the steps of: vaccinating said individual with said SCCE orSCCE peptide; isolating T cells from said individual; and measuringimmune responses induced by said SCCE or SCCE peptide, wherein anincreased level of immune responses compared to those exhibited by cellsfrom normal individual indicates that said individual has beenvaccinated by said SCCE or SCCE peptide.
 2. The method of claim 1,wherein said immune response is selected from the group consisting of Tcell proliferation induced by said SCCE or SCCE peptide, frequency ofcytokine-secreting T cells specific to said SCCE or SCCE peptide andfrequency of T cells expressing T cell receptor specific to said SCCE orSCCE peptide.
 3. The method of claim 1, wherein said SCCE peptide isselected from the group consisting of SEQ ID Nos. 31, 32, 33, 34, 35,36, 80, 86 and
 99. 4. A method of inhibiting stratum corneum chymotryticenzyme protein in a cell, comprising the step of: introducing into saidcell an antibody specific for a stratum corneum chymotrytic enzyme(SCCE) protein or a fragment thereof, wherein binding of said antibodyto said stratum corneum chymotrytic enzyme protein inhibits said stratumcorneum chymotrytic enzyme protein in said cell.
 5. The method of claim4, wherein said SCCE protein fragment is selected from the groupconsisting of SEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86 and
 99. 6. Amethod of targeted therapy to an individual, comprising the step of:administering a compound to an individual, wherein said compound has atherapeutic moiety and a targeting moiety specific for stratum corneumchymotrytic enzyme.
 7. The method of claim 6, wherein said targetingmoiety is selected from the group consisting of an antibody specific forstratum corneum chymotrytic enzyme and a ligand or ligand binding domainthat binds stratum corneum chymotrytic enzyme.
 8. The method of claim 6,wherein said therapeutic moiety is a radioisotope, a toxin, achemotherapeutic agent, an immune stimulant, or a cytotoxic agent. 9.The method of claim 6, wherein said individual suffers from a diseaseselected from the group consisting of ovarian cancer, lung cancer,prostate cancer, pancreatic cancer and colon cancer.
 10. An immunogeniccomposition, comprising an immunogenic fragment of a stratum corneumchymotrytic enzyme protein and an appropriate adjuvant.
 12. Theimmunogenic composition of claim 10, wherein the length of said stratumcorneum chymotrytic enzyme fragment is from about 9-residue long toabout 20-residue long.
 13. The immunogenic composition of claim 11,wherein said 9-residue fragment is selected from the group consisting ofSEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86 and
 99. 14. Anoligonucleotide having a sequence complementary to SEQ ID No.
 30. 15. Acomposition comprising the oligonucleotide of claim 14 and aphysiologically acceptable carrier.
 16. A method of treating aneoplastic state in an individual in need of such treatment, comprisingthe step of: administering to said individual an effective dose of theoligonucleotide of claim
 15. 17. The method of claim 16, wherein saidneoplastic state is selected from the group consisting of ovariancancer, breast cancer, lung cancer, colon cancer, prostate cancer,pancreatic cancer and other cancers in which stratum corneum chymotryticenzyme is overexpressed.
 18. A method of inhibiting expression ofendogenous SCCE in a cell, comprising the step of: introducing into saidcell a vector comprising the oligonucleotide of claim 14, whereinexpression of said vector in said cell produces SCCE antisense RNA thathybridizes to endogenous SCCE mRNA, thereby inhibiting expression ofendogenous SCCE in said cell.